Activating mutations in Protein Tyrosine Phosphatase, non-receptor type 11 (PTPN11) are seen in approximately 5-10% percent of patients with acute myeloid leukemia (AML) that independently confers a poor prognosis, and is the leukemic driver in 35% of patients with the rare chemoresistant juvenile myelomonocytic leukemia (JMML). To date, targeting PTPN11 for clinical benefit has not been well established. Our preliminary data using high-throughput functional assays have identified that PTPN11 activity is dependent on an upstream tyrosine kinase, TNK2, and that leukemic cells with activating PTPN11 mutants are sensitive to TNK2 inhibition. The long-term goal of this proposal is to identify novel combinations of targeted therapies using PTPN11/TNK2 dependent leukemias as a model. The immediate goals are to comprehensively understand the molecular mechanisms of PTPN11 activation, and to define therapeutic regimens for treatment based on the central hypothesis that PTPN11-dependent leukemias are directly activated by TNK2. To accomplish these goals, several key AIMS will need to be addressed: 1) Develop a detailed understanding of the biochemical pathways interacting with activated PTPN11. We will use molecular and biochemical studies to dissect the interactions of proteins within PTPN11/TNK2 and downstream pathways through heterologous model systems, PTPN11 mutant AML cell lines, murine bone marrow cells with mutant PTPTN11, and primary mutant PTPN11 AML samples. Further, RNAi and CRISPR technology will be utilized to selectively inhibit expression of candidate proteins to identify critical interactions necessary for PTPN11/TNK2 activity within hematopoietic model systems. These studies will establish the molecular and biochemical foundation necessary for defining PTPN11/TNK2 activation, and help guide novel targeted approaches. 2) Test combination pathway inhibition to overcome escape from monotherapy. We will use our experience and expertise with small molecule inhibitors to identify synergistic combinations of inhibitors to TNK2, PTPN11, modulators of PTPN11/TNK2 activation, and downstream effectors in vitro using cell lines and primary patient samples. Molecular and biochemical studies will be performed on samples that overcome therapy to identify escape mechanisms. These studies will define the use of combination therapy for treatment of PTPN11-driven leukemias and provide critical pre-clinical data for further development of these combinations. 3) Test combination therapies in the animal model. We will test the above combinations in vivo using mouse models harboring PTPN11 mutations, as well as primary patient leukemia samples with PTPN11 mutations xenografted into immunodeficient mice. These studies will build the preclinical foundation for combination of targeted therapies for eventual use in patients with PTPN11-driven leukemias.